Biology subjects

Reinke, A. W.

Publications and source records attributed to Reinke, A. W..

At least 19 recordsLinked to original sources

bioRxiv2026

An expanded microsporidia species attribute database documents the extensive ecological and phenotypic diversity of these parasites

Microsporidia are a group of parasites that exhibit vast morphological, ecological, and genetic diversity. To catalog this diversity, we previously generated a database of attributes for microsporidia species. Here we expand this database to include species described since 2021, previously missed descriptions, and provisional species defined by clustering GenBank 18S rRNA sequences, resulting in 1,795 species. Using these data, we describe when and where microsporidia have been reported, their hosts and host habitats, the tissues they infect, and their spore morphology. A phylogeny of 18S sequence clusters shows that closely related microsporidia tend to infect closely related hosts, such that sequence identity is predictive of host taxonomy. Clustering also reveals previously unappreciated genetic diversity within some named species, in particular the human pathogen Vittaforma corneae, suggesting that these may comprise multiple species. We have also generated a web portal through which new species information can be submitted, which will facilitate regular updates. This database provides a resource for systematic analysis of microsporidia properties and a reference for taxonomic descriptions.

microbiology↗

bioRxiv2026

Most inhibitors with defined yeast targets have limited activity against the microsporidian Nematocida parisii

Microsporidia are obligate intracellular parasites that infect diverse animals. Budding yeast has long been used to define core molecular pathways and inhibitors that target them. To test whether these compounds can target conserved pathways in the microsporidian Nematocida parisii, we assessed 15 inhibitors with defined yeast targets that had not previously been tested for effects on infection of Caenorhabditis elegans. Most showed little activity against N. parisii, except tunicamycin. These results identify tunicamycin as a potential tool for studying endoplasmic reticulum stress in microsporidia, while indicating that most yeast-targeted inhibitors are largely ineffective in this system.

microbiology↗

bioRxiv2026

Forward genetics reveals microsporidian drug targets and recombination-based resistance

Microsporidia are a diverse group of intracellular fungal parasites that infect humans and many agriculturally important animals. Several microsporidia inhibitors have been characterized, but the targets of these inhibitors in microsporidia have not been confirmed, and the mechanisms by which microsporidia evolve resistance are unknown. Using the model organism Caenorhabditis elegans and its naturally occurring microsporidian parasite Nematocida parisii, we developed a technique to continually passage infected animals for multiple generations under increasing inhibitor concentrations. We obtained multiple independent N. parisii strains that are specifically resistant to the microsporidia inhibitors albendazole or dexrazoxane. The albendazole-resistant strains all contained either heterozygous or homozygous mutations in beta-tubulin, with several strains containing beta-tubulin variants that have been observed in other albendazole-resistant organisms. Strains containing homozygous variants are resistant to several albendazole analogs, whereas the heterozygous variant-containing strains are only resistant to albendazole. Several of the albendazole-resistant strains also contain mutations in alpha-tubulin. The dexrazoxane-resistant strains all contain heterozygous mutations in topoisomerase II and several of these mutations occur in the binding site of this inhibitor. By mapping heterozygosity of the resistant strains across the N. parisii genome, we observed loss of heterozygosity spanning the beta-tubulin locus in the albendazole-resistant strains containing homozygous beta-tubulin variants. Our study demonstrates the utility of forward genetics for identifying microsporidian drug targets. We also find that drug resistance arises through de novo heterozygous mutations that can subsequently become homozygous, likely via mitotic recombination.

microbiology↗

bioRxiv2025

Worms About Town: a citizen science outreach project discovers microsporidian parasites of nematodes through environmental sampling

Environmental sampling of animal species can be a powerful tool for understanding aspects of host-parasite systems like species diversity and mechanisms of host specificity. To this end, we developed a citizen science project called Worms About Town to survey wild nematodes across Toronto, Ontario for microsporidia infection, which are natural fungal parasites of nematodes. Science communication and learning were emphasized through in-person outreach events and two-way discussion of project findings. Participants were equipped with tailored nematode collection kits to collect rotten plant substrates from local gardens and parks. In the first year of the project (2024), 13 participants acquired 127 samples, 60 of which contained nematodes and four were infected with microsporidia parasites. Microsporidia-infected nematodes were discovered from multiple types of plant substrates from various locations across Toronto. Sequencing of the microsporidia 18S rRNA gene of infected hosts identified two isolates of Nematocida homosporus, which is a known generalist microsporidian parasite of nematodes. The other parasites found in this study are undescribed species belonging to the Pancytospora and Enteropsectra genera, two microsporidia taxa that are distantly related to a clade of human-infecting microsporidia and known to infect wild nematodes. Three nematode isolates of the Caenorhabditis genus were discovered, including two isolates of Caenorhabditis elegans, a species not previously reported in Ontario. Both of the N. homosporus isolates were found in C. elegans, and one can infect a laboratory strain of C. elegans. The Worms About Town project will organize repeated wild nematode collections each year to determine natural factors that impact microsporidia infection in nematodes and to further characterize the diversity of this host-parasite system.

microbiology↗

bioRxiv2025

Identification of natural products and synthetic analogs which inhibit microsporidia spores and prevent infection

Microsporidiosis, a disease caused by microsporidia, affects many animals, with symptoms ranging from diarrhea to death, especially in immunocompromised individuals. Current treatments, such as albendazole and fumagillin, are limited in efficacy. To address this problem, we utilized Caenorhabditis elegans infected with its natural microsporidian Nematocida parisii to evaluate 4,080 structurally diverse compounds from the Boston University Center for Molecular Discovery (BU-CMD) chemical library. From this screen we identified 34 compounds that effectively inhibited N. parisii infection and restored the reproductive capacity of C. elegans. All 17 compounds we validated prevented N. parisii infection in C. elegans, and 10 were capable of suppressing microsporidia invasion by inactivating mature spores. Additionally, five of the identified compounds were also effective against Pancytospora epiphaga, a species related to human-infecting microsporidia. Together this work identifies and characterizes compounds which inhibit microsporidia infection. ImportanceMicrosporidia are a large group of microbial parasites that infect many animals, including humans. Many agriculturally important animals such as honey bees, shrimp, crabs, and fish are infected by microsporidia, and these infections often result in smaller animals, fewer offspring, and death. Only two drugs are commonly used to treat microsporidia infections; due to some species being resistant and host toxicity there is a need to identify other microsporidia inhibitors. In this study, we screened over 4000 small molecules using a model round worm infected with microsporidia. We identified several dozen inhibitors and characterized how these compounds prevented infection by determining which stage of the parasite they act upon. Together our work identified and characterized compounds that could be used as a starting point to design better microsporidia inhibitors.

microbiology↗

bioRxiv2025

Small-molecule screen in C. elegans identifies benzenesulfonamides as inhibitors of microsporidia spores

Microsporidia, a large group of fungal-related intracellular parasites, infect several economically significant animals, leading to substantial economic losses. As currently available anti-microsporidia therapies are either ineffective or come with numerous adverse effects, there is a need for alternative microsporidia inhibitors. Here we screen a subset of the ChemBridge DIVERset library, comprising 2500 diverse compounds, using Caenorhabditis elegans infected with its natural microsporidian parasite, Nematocida parisii. By testing these compounds at 60 M in 96-well assay plates, we identified 26 hits that restored the ability of C. elegans to produce progeny in the presence of N. parisii. We confirmed that out of 20 tested compounds, 18 ChemBridge compounds effectively inhibit N. parisii infection in C. elegans. Of these 18, 10 were benzenesulfonamide derivatives which inhibit microsporidia infection by inactivating spores. We screened an additional 475-compound benzenesulfonamide library, successfully identifying three compounds that are effective at a lower concentration than the initial hits. We further show that one benzenesulfonamide compound displays inhibitory activity against several species of microsporidia, inhibiting infection of species belonging to the Nematocida, Enterocytozoon, and Encephalitozoon genera. Together our results suggest that benzenesulfonamides are a potential scaffold for the development of microsporidia antiseptics.

microbiology↗

bioRxiv2025

Microsporidia infection alters C. elegans lipid levels

Microsporidia are fungal-related obligate intracellular parasites that infect many types of animals. Microsporidia have exceptionally reduced genomes resulting in limited metabolic capabilities and are thought to be reliant on host metabolism to fuel their own growth. Here, we investigate the impact of microsporidia infection on host lipid metabolism using the nematode Caenorhabditis elegans along with its natural microsporidian pathogen Nematocida parisii. We show that infection causes an increase in the level of C. elegans lipid droplet associated lipase, ATGL-1, and a decrease in host fat levels. A mutation that decreases ATGL-1 activity and overexpression of ATGL-1 did not significantly change N. parisii infection levels. Using lipidomics we show that N. parisii infection decreases C. elegans triglyceride levels and results in increased ceramides that we speculate are synthesized by N. parisii. Mutations in host genes involved in ceramide synthesis did not significantly change the levels of N. parisii infection. Together these results show that microsporidia can cause changes to lipid metabolism of their hosts, but some individual mutations of C. elegans lipid enzymes do not alter microsporidian growth.

microbiology↗

bioRxiv2024

The Caenorhabditis elegans bacterial microbiome influences microsporidia infection through nutrient limitation and inhibiting parasite invasion

Microsporidia are eukaryotic obligate intracellular parasites that infect most animals including humans. To understand how the microbiome can impact microsporidia infection, we tested how bacterial isolates that naturally occur with Caenorhabditis elegans influence infection by the microsporidian Nematocida parisii. Nematodes exposed to two of these bacteria, Chryseobacterium scopthalmum and Sphingobacterium multivorum, exhibit reduced pathogen loads. Using untargeted metabolomics, we show that unsaturated fatty acid levels are disrupted by growth on these bacteria and that supplementation with the polyunsaturated fatty acid linoleic acid can restore full parasite growth in animals cultured on S. multivorum. We also found that two isolates, Pseudomonas lurida and Pseudomonas mendocina, secrete molecules that inactivate N. parisii spores. We determined that P. lurida inhibits N. parisii through the production of massetolides. We then measured 53 additional Pseudomonas strains, 64% of which significantly reduced N. parisii infection. A mixture of Pseudomonas species can greatly limit the amount of infection in C. elegans populations over many generations. Our findings suggest that interactions between bacteria and N. parisii are common and that these bacteria both modulate host metabolism and produce compounds that inhibit microsporidia infection.

microbiology↗

bioRxiv2024

Functional consequences of reductive protein evolution in a minimal eukaryotic genome

Microsporidia are parasites with the smallest known eukaryotic genomes. The extent of protein loss in these organisms has been well documented, but much less is known about how compaction of microsporidia proteins affects their function. Taking a comparative genomic approach, we identified microsporidia orthologs of budding yeast proteins and show that these orthologs are enriched for essential yeast genes. We show that the median microsporidia protein is 21% shorter than its yeast counterpart and although extensive protein loss occurred after the divergence of microsporidia, reduced protein sizes were already present in microsporidian relatives. Microsporidia proteins are shorter through reduced domain lengths, diminished linker lengths, and domain loss, with 21% of microsporidia orthologs having lost domains present in yeast. On average, 34% of microsporidia orthologs have lost C-terminal residues essential for function in yeast, including 13 essential domains lost per genome. We also found that microsporidia display distinct phylogenetic patterns of domain loss, with losses occurring in a clade-specific manner. To investigate conservation of function, we used yeast complementation assays to test orthologs from several microsporidia species and their relative Rozella allomycis. These experiments reveal that most microsporidia proteins cannot complement their yeast orthologs, the ability to complement is about three-fold less than observed for R. allomycis orthologs, and proteins that do not complement are more reduced in length than their yeast orthologs. Altogether, our results demonstrate the drastic reduction of microsporidia proteins and show that these reductions have resulted in functional divergence from their fungal ancestors.

evolutionary biology↗

bioRxiv2023

Parental dietary vitamin B12 causes intergenerational growth acceleration and protects offspring from pathogenic microsporidia and bacteria

The parental environment of C. elegans can have lasting effects on progeny development and immunity. Vitamin B12 exposure in C. elegans has been shown to accelerate development and to reduce killing caused by pathogenic Pseudomonas aeruginosa. Vitamin B12 is also maternally transferred to offspring, suggesting a potential role in progeny development. However, the intergenerational effects of vitamin B12 in C. elegans remain uncharacterized. Here, we show that parental exposure to dietary vitamin B12 or vitamin B12-producing bacteria results in offspring with accelerated growth. We find that this inherited acceleration phenotype is vitamin B12 dose-dependent and persists for a single generation. Progeny from vitamin B12 treated parents display reduced levels of the acdh-1 dietary sensor in the earliest larval stages, with expression of this protein recovering into adulthood. During infection with Nematocida parisii, a natural microsporidian pathogen, the offspring of worms fed vitamin B12 diets have better reproductive fitness. However, vitamin B12 diet does not affect offspring infection levels, suggesting that accelerated development provides tolerance to microsporidian infection. Offspring from parents exposed to vitamin B12 are also protected from killing by pathogenic Pseudomonas vranovensis. Vitamin B12-induced intergenerational growth acceleration and N. parisii tolerance is dependent upon the methionine biosynthesis pathway. However, protection from P. vranovensis killing is mediated through both the methionine biosynthesis and the propionyl-CoA breakdown pathways. Our results show how parental microbial diet impacts progeny development through the transfer of vitamin B12 which results in accelerated growth and pathogen tolerance.

genetics↗

bioRxiv2023

Screening of the Pandemic Response Box identifies anti-microsporidia compounds

Microsporidia are fungi obligate intracellular pathogens, which infect most animals and cause microsporidiosis. Despite the serious threat that microsporidia pose to humans and agricultural animals, few drugs are available for the treatment and control of microsporidia. To identify novel inhibitors, we took advantage of the model organism Caenorhabditis elegans infected with its natural microsporidian Nematocida parisii. We used this system to screen the Pandemic Response Box, a collection of 400 diverse compounds with known antimicrobial activity. After testing these compounds in a 96-well format at high (100 M) and low (40 M) concentrations, we identified four inhibitors that restored the ability of C. elegans to produce progeny in the presence of N. parisii. All four compounds reduced the pathogen load of both N. parisii and Pancytospora epiphaga, a C. elegans-infecting microsporidia related to human-infecting species. One of these compounds, a known inhibitor of a viral protease, MMV1006203, inhibited invasion and prevented the firing of spores. A bis-indole derivative, MMV1593539, decreased spore viability. An albendazole analog, MMV1782387, inhibited proliferation of N. parisii. We tested albendazole as well as 5 other analogs and observed that MMV1782387 was amongst the strongest inhibitors of N. parisii and displayed the least host toxicity. Our study further demonstrates the effectiveness of the C. elegans-N. parisii system for discovering microsporidia inhibitors and the compounds we identified provide potential scaffolds for anti-microsporidia drug development.

microbiology↗

bioRxiv2022

Genomic and phenotypic evolution of nematode-infecting microsporidia

Microsporidia are a large phylum of intracellular parasites that can infect most types of animals. Species in the Nematocida genus can infect nematodes including Caenorhabditis elegans, which has become an important model to study mechanisms of microsporidia infection. To understand the genomic properties and evolution of nematode-infecting microsporidia, we sequenced the genomes of nine species of microsporidia, including two genera, Enteropsectra and Pancytospora, without any previously sequenced genomes. Core cellular processes, including metabolic pathways, are mostly conserved across genera of nematode-infecting microsporidia. Each species encodes unique proteins belonging to large gene families that are likely used to interact with host cells. Most strikingly, we observed one such family, NemLGF1, is present in both Nematocida and Pancytospora species, suggesting horizontal gene transfer between species from different genera. To understand how Nematocida phenotypic traits evolved, we measured the host range, tissue specificity, spore size, and polar tube length of several species in the genus. Our phylogenetic analysis shows that Nematocida is composed of two groups of species with distinct traits and that species with longer polar tubes infect multiple tissues. Together, our work details both genomic and trait evolution between related microsporidia species and provides a useful resource for further understanding microsporidia evolution and infection mechanisms. Author SummaryMicrosporidia are microbial parasites that can infect many animals. Nematodes have become a useful system to study microsporidia as these animals are commonly infected by microsporidia and these infections can be easily studied in a laboratory environment. To better understand how microsporidia evolve and change their properties as they infect different hosts, we sequenced the genomes of nine microsporidia species which infect nematodes. We found that metabolic pathways were mostly conserved between different clades of these species. Surprisingly, we found a family of proteins predicted to facilitate host interactions that is present in two distinct genera, suggesting that these genes were transferred between microsporidia species. We also determined the hosts and tissues that these microsporidia can infect as well as morphological properties of the microsporidia. We show that these properties are quite variable in related microsporidia species. Our results provide insight into the evolution of microsporidia and also provide a resource for studying microsporidia infections in nematodes.

microbiology↗

bioRxiv2022

A Comprehensive Survey of C. elegans Argonaute Proteins Reveals Organism-wide Gene Regulatory Networks and Functions

Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode C. elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work we systematically analyzed every C. elegans AGO, using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.

molecular biology↗

bioRxiv2022

High-throughput phenotyping of C. elegans wild isolates reveals specific resistance and susceptibility traits to infection by distinct microsporidia species

Animals are under constant selective pressure from a myriad of diverse pathogens. Microsporidia are ubiquitous animal parasites, but the influence they exert on shaping animal genomes is mostly unknown. Using multiplexed competition assays, we measured the impact of four different species of microsporidia on 22 wild isolates of Caenorhabditis elegans. This resulted in the identification and confirmation of 13 strains with significantly altered population fitness profiles under infection conditions. One of these identified strains, JU1400, is sensitive to an epidermal-infecting species by lacking tolerance to infection. JU1400 is also resistant to an intestinal-infecting species and can specifically recognize and destroy this pathogen. Genetic mapping of JU1400 demonstrates that these two opposing phenotypes are caused by separate loci. Transcriptional analysis reveals the JU1400 sensitivity to epidermal microsporidia infection results in a response pattern that shares similarity to toxin-induced responses. In contrast, we do not observe JU1400 intestinal resistance being regulated at the transcriptional level. The transcriptional response to these four microsporidia species is conserved, with C. elegans strain-specific differences in potential immune genes. Together, our results show that phenotypic differences to microsporidia infection amongst C. elegans are common and that animals can evolve species-specific genetic interactions.

genomics↗

bioRxiv2022

Conservation of Nematocida microsporidia gene expression and host response in Caenorhabditis nematodes

Microsporidia are obligate intracellular parasites that are known to infect most types of animals. Many species of microsporidia can infect multiple related hosts, but it is not known if microsporidia express different genes depending upon which host species is infected or if the host response to infection is specific to each microsporidia species. To address these questions, we took advantage of two species of Nematocida microsporidia, N. parisii and N. ausubeli, that infect two species of Caenorhabditis nematodes, C. elegans and C. briggsae. We performed RNA-seq at several time points for each host infected with either microsporidia species. We observed that Nematocida transcription was largely independent of its host. We also observed that the host transcriptional response was similar when infected with either microsporidia species. Finally, we analyzed if the host response to microsporidia infection was conserved across host species. We observed that although many of the genes upregulated in response to infection are not direct orthologs, the same expanded gene families are upregulated in both Caenorhabditis hosts. Together our results describe the transcriptional interactions of Nematocida infection in Caenorhabditis hosts and demonstrate that these responses are evolutionarily conserved. Significance statementMicrosporidia are a powerful model to study pathogen evolution, but much is still unknown about how these pathogens have evolved to infect multiple host species. We found that microsporidia express most of their genes similarly even when they are infecting different host species and that related host species respond similarly to different microsporidia. Our results suggests that there are conserved transcriptional responses during microsporidia infection.

genomics↗

bioRxiv2021

High-throughput small molecule screen identifies inhibitors of microsporidia invasion and proliferation in C. elegans.

Microsporidia are a diverse group of fungal-related obligate intracellular parasites that infect most animal phyla. Despite the emerging threat that microsporidia have become to humans and agricultural animals, few reliable treatment options exist. To identify novel chemical inhibitors of microsporidia infection, we developed a high-throughput screening method using Caenorhabditis elegans and the microsporidia species Nematocida parisii. We screened the Spectrum Collection of 2,560 FDA-approved compounds and natural products to identify compounds that prevent C. elegans progeny inhibition caused by N. parisii infection. We developed a semi-automated method for quantifying C. elegans progeny number in liquid culture, confirming 11 candidate microsporidia inhibitors. We show that five compounds prevent microsporidia infection by inhibiting spore firing, and demonstrate that one compound, dexrazoxane, slows infection progression. We also show that these compounds have activity against several other microsporidia species, including those which infect humans. Together, our results demonstrate the effectiveness of C. elegans as a model host for drug discovery against intracellular pathogens and provide a scalable high-throughput system for the identification and characterization of additional microsporidia inhibitors.

microbiology↗

bioRxiv2021

An intestinally secreted host factor limits bacterial colonization but promotes microsporidia invasion of C. elegans

Microsporidia are ubiquitous obligate intracellular pathogens of animals. These parasites often infect hosts through an oral route, but little is known about the function of host intestinal proteins that facilitate microsporidia invasion. To identify such factors necessary for infection by Nematocida parisii, a natural microsporidian pathogen of Caenorhabditis elegans, we performed a forward genetic screen to identify mutant animals that have a Fitness Advantage with Nematocida (Fawn). We isolated four fawn mutants that are resistant to Nematocida infection and contain mutations in T14E8.4, which we renamed aaim-1 (Antibacterial and Aids invasion by Microsporidia). Expression of AAIM-1 in the intestine of aaim-1 animals restores N. parisii infectivity and this rescue of infectivity is dependent upon AAIM-1 secretion. N. parisii spores in aaim-1 animals are improperly oriented in the intestinal lumen, leading to reduced levels of parasite invasion. Conversely, aaim-1 mutants display both increased colonization and susceptibility to the bacterial pathogen Pseudomonas aeruginosa and overexpression of AAIM-1 reduces P. aeruginosa colonization. Competitive fitness assays show that aaim-1 mutants are favoured in the presence of N. parisii but disadvantaged on P. aeruginosa compared to wild type animals. Together, this work demonstrates how microsporidia exploits a secreted protein to promote host invasion. Our results also suggest evolutionary trade-offs may exist to optimizing host defense against multiple classes of pathogens.

microbiology↗

bioRxiv2021

Intergenerational adaptations to stress are evolutionarily conserved, stress-specific, and have deleterious trade-offs

Despite reports of parental exposure to stress promoting physiological adaptations in progeny in diverse organisms, there remains considerable debate over the significance and evolutionary conservation of such multigenerational effects. Here, we investigate four independent models of intergenerational adaptations to stress in C. elegans - bacterial infection, eukaryotic infection, osmotic stress and nutrient stress - across multiple species. We found that all four intergenerational physiological adaptations are conserved in at least one other species, that they are stress-specific, and that they have deleterious trade-offs in mismatched environments. By profiling the effects of parental bacterial infection and osmotic stress exposure on progeny gene expression across species we established a core set of 279 highly conserved genes that exhibited intergenerational changes in expression in response to stress in all species tested and provide evidence suggesting that presumed adaptive and deleterious intergenerational effects are molecularly related at the gene expression level. By contrast, we found that these same stresses did not elicit any similarly conserved transgenerational changes in progeny gene expression three generations after stress exposure. We conclude that intergenerational responses to stress play a substantial and evolutionarily conserved role in regulating animal physiology and that the vast majority of the effects of parental stress on progeny gene expression are reversible and not maintained transgenerationally.

genetics↗